The Human Paraoxonase 2: An Optimized Procedure for Refolding and Stabilization Facilitates Enzyme Analyses and a Proteomics Approach

Abstract

The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.

Keywords: biofilm; enzyme kinetics; lactonase; paraoxonase; quorum quenching.

Figure 1

Comparison between the previously reported purification procedure of rPON2 according to Mandrich et al. [20] (on the (left side)) and the new purification procedure reported in this work (on the (right side)).

Figure 2

rPON2 purification analysis by SDS-PAGE. Panel (a), lane 1: total IPTG-induced cell lysate. Lane 2: supernatant after centrifugation. Lane 3: Triton-X100 wash of pelleted inclusion bodies. Lane 4: total inclusion bodies. Lane 5: solubilized inclusion bodies. Lane 6: non-solubilized inclusion bodies. Panel (b), lane M: molecular weight markers. Lanes 7–11: fractions eluted from Ni-NTA column. Panel (c), lane M: molecular weight markers. Lane 12: purified protein after gel filtration (see Figure 3).

Figure 3

(a) Size-exclusion chromatography purification step of rPON2 on 26/600 Superdex G-75 column. Protein was assessed by absorbance reading at 280 nm (continuous trace) and by activity measurements with the substrate 3oxoC12-HSL (dotted trace); (b) Native PAGE highlighting the presence of a multimer–monomer equilibrium. F7 represents the fraction corresponding to the first peak (Peak 1) of size-exclusion chromatography, while F16 to F21 are the fractions corresponding to the second peak (Peak 2).

Figure 4

Far-UV CD spectrum of rPON2 (0.1 mg/mL) in a 0.1 cm quartz cuvette. Data are buffer subtracted, normalized for protein concentration, and reported as mean residue ellipticity. In the inset, the secondary structure content estimation is reported, which was performed with the webserver BestSel v1.3.230210.

Figure 5

rPON2 residual activity over time (days), expressed as a percentage of the initial activity at day 0. Hydrolysis of pNP-C3 was monitored at 405 nm in 96-well plates, by using a multiplate reader. The chemical additives used were NaCl; trehalose; glycerol; Triton X-100; and ethylene glycol.

Figure 6

rPON2 residual activity over time (days), expressed as a percentage of the initial activity at day 0. Lactonase activity on 3oxoC12-HSL for the protein stored at 4 °C in the buffer only (blue) and at −20 °C in buffer with the addition of 20% glycerol (orange).

Figure 7

Michaelis–Menten curve of rPON2 for 3oxoC12-HSL at 37 °C (25–500 µM).

Figure 8

Biofilm inhibition capacity of rPON2, at the reported concentrations. The biofilm formation is expressed as a percentage of the biofilm formation compared to the control PAO1 cells with protein buffer, quantified by the crystal violet assay.

Figure 9

PON2 interactome. Interactions were predicted based on the String database (version 12.0). Known interactions (from curated databases and experimentally determined) are marked by pink and light blue lines and predicted interactions (gene neighborhood, gene fusion, and gene co-occurrence) are marked by green, red, and blue lines, respectively. The interactions that we identified commonly in multiple experiments with PON2 are shown by the red dotted lines.

Figure 10

Sestrin 2 role in renal oxidative stress as modified from [47], and dopamine D2 receptor (D2R) upregulates Sestrin 2, reducing renal oxidative stress and maintaining normal blood pressure. D2R activation positively affects PON2, which, when active, inhibits NADPH oxidase and boosts Sestrin 2 expression. This dual action aids in reducing hyperoxidized peroxiredoxins (Prxs), effectively mitigating renal oxidative stress and ensuring normal blood pressure.